Electrophoresis involves the separation of charged macromolecular species in a carrier medium in an electric field. This involves the migration of charged molecular species through a porous gel under an applied electric field. Commonly used gels include polyacrylamides, typically crosslinked with a small amount of bis-acrylamide, and other similar gels.
Electrophoresis gels are often held in place by a cassette during use. Examples of gel electrophoresis devices are shown in U.S. Pat. Nos. 4,909,918 and 4,975,174 which patents are hereby incorporated by reference in their entirety.
One useful application of electrophoresis gel separation technology has been in the area of lipoprotein separations. As is known, cardiovascular disease is the leading cause of death in the developed world, accounting either directly or indirectly, for half of all deaths and consuming the largest single portion of health care costs. It is also generally known that cardiovascular disease is influenced at least indirectly, by the lipoprotein levels within the blood.
With this understanding in mind, there has been much effort expended in the area of lipoprotein separation. The early advancements in lipoprotein separations were performed by density gradient ultracentrifugation and the lipoproteins were found to consist of chylomicra, very low density lipoproteins (VLDL), intermediate density lipoproteins (IDL), low density lipoproteins (LDL), and high density lipoproteins (HDL). These subfractions were verified by for example, cellulose acetate and acrylamide electrophoresis, with agarose and acrylamide demonstrating that there was a precise inverse order between lipoprotein density and molecular size.
It has also been found that abnormally elevated LDL blood levels correlate with elevated cardiovascular disease risk. It is has been conversely found that elevated HDL levels correlate with increased cardiovascular health. More importantly, LDL and HDL are inversely co-dependant, and recent transgenic and "knock out" protocols have demonstrated a causal link in the co-dependency.
Improvements in centrifugation, the use of antibodies and improvements in electrophoresis led to further subfraction and functional analysis of the chylomicra, VLDL, LDL and HDL families of lipoproteins. The LDL family (LDLF) was found to contain four variations by centrifugation and seven variations by electrophoresis. In general, heavy LDLs are suggested as being strongly related to elevated cardiovascular disease risk.
Similarly, the HDL lipoprotein subfractions fall under three major classes, known as HDL.sub.1, HDL.sub.2, and HDL.sub.3 ; with HDL.sub.1 being the largest and least dense, HDL.sub.2 being intermediate and HDL.sub.3 being the smallest and most dense. The HDL.sub.1 group has been further divided into HDL.sub.1a, HDL.sub.1b and HDL.sub.1c. The HDL.sub.1 group is the only HDL group know to deliver lipids to tissues and the liver via protein receptor systems. The other HDL families are powerful lipid scavengers with HDL.sub.2 being more efficient than the HDL.sub.3. There appears to be a very strong correlation between HDL.sub.2 and HDL.sub.3 levels in cardiovascular disease risk. All of the HDLs can be collectively referred to as the HDL family (HDLF).
All of the lipoprotein subfractions appear to be regulated by discrete, large mass transfers, and an ability to monitor the subfraction quantities is critical to obtaining a precise understanding of the system, and subsequently, accurate diagnosis.
With the advent of acrylamide gel electrophoresis, it was possible to achieve separations which could match the resolutions of ultracentrifugation techniques. Quantitative equivalency to ultra centrifugation was established. With the introduction of gradient gels, based upon the concentration of the polyacrylamide within the various gradients, it was possible to surpass the resolving power of ultracentrifugation in the separation of both LDL subfractions and HDL subfractions.
There are at least two problems that occur with the use of high resolution gradient gels. First, heretofore known gels separate macromolecules on the basis of size and charge. If charge is a constant portion of molecular size in a dispersed macromolecular population, small molecules are separated predominantly by charge while large molecules closer to the pore size exclusion limit of the gel, are separated primarily on the basis of size. In a gradient gel, higher resolution is achieved as molecules are progressively separated on the basis of mass as the run time continues, regardless of charge differences. Ultimately, molecular mobility is halted as the macromolecules achieve their pore size exclusion limits. To reach pore size exclusion limits, or equilibrium, very long electrophoresis run times are necessary. If the macromolecules are very soluble, very high resolution can be achieved in short, nonequalibrium run times. If the macromolecules are insoluble, they cannot be electrophoresed at all. In standard, non-denaturing electrophoresis buffers of common use, HDLs are very soluble, LDLs are moderately soluble and VLDLs and IDLs are moderately to very insoluble.
A need exists therefore for an electrophoresis system which can simultaneously monitor the normal and abnormal amounts, types and compositions of lipoproteins, in a timely manner.